DESCRIPTION: Human beta defensins (hBDs), small, cationic, antimicrobial, innate response molecules expressed in mucosal epithelia, function as a first line of host defense against microbial pathogenesis. We have shown that oral epithelium and cells not only express the constitutive hBD1 and the inducible hBD2 and -3, but that the latter two are always "on" in normal intact oral epithelium. This is in stark contrast to other mucosae of the body where inflammation or infection induces their expression. Our data suggest that homeostatic hBD expression may be due to the exposure of the oral tissue to specific hBD-inducing commensal bacteria. Fusobacterium nucleatum, a ubiquitous Gram negative bacterium of the oral cavity, induces hBD-2 and -3 expression in normal human oral epithelial cells (NHOECs), while Porphyromonas gingivalis, a major Gram negative opportunist involved in the initiation of periodontal disease, and which efficiently invades NHOECs and oral tissues, does not. Our work has led us to establish a model showing that F. nucleatum challenged human oral epithelial cells are protected from Porphyromonas gingivalis invasion. We posit that this protection is elicited through induction of hBDs. Interestingly, F. nucleatum is resistant to hBD1, -2 and -3, while P. gingivalis is sensitive to all three agents in low micromolar concentrations. Since the role of hBDs in protecting the oral mucosal epithelium has not been systematically studied, nor has serious consideration been given to differential expression of these and other NHOEC derived antimicrobial peptides or the mechanisms by which hBD protection is elicited, we propose the following Aims: (I) Determine if inhibiting the production of hBD2 or hBD3 renders NHOECs susceptible to P. gingivalis invasion following F. nucleatum activation, (II) Use a subtractive proteomics approach to determine the protein differences in NHOECs challenged with F. nucleatum versus P. gingivalis and (HI) Resolve whether soluble hBD is necessary for protection of NHOECs following bacterial challenge. With our i) working model of bacterially challenged NHOEC monolayers in place, ii) ability to block hBDs in oral epithelial cells through small inhibitory RNA strategies, iii.) ability to produce recombinant hBDs, and iv) capabilities in conducting proteomics based studies, we will be able to investigate further the role these peptides play in defense and steady state conditions of the oral cavity. Studies proposed herein could lead to a better understanding of the mechanisms through which hBDs function and their contribution to the maintenance of localized mucosal health.